Critical electrostatic F-actin-Tm and F-actin-Tm-myosin interactions. (A) Molecular models showing the location of tropomyosin (Tm) (blue) on actin (yellow and green) in the absence and presence of the myosin head (S1) (red) bound in rigor. The F-actin-Tm and rigor F-actin-Tm-myosin structures are based on those generated by Li et al. () and Behrmann et al. () respectively. (B) Enlarged views illustrate critical electrostatic associations between actin and Tm in the absence or presence of S1. K328 on actin (circled) interacts with E181 of Tm in the absence of myosin (left) and with E286 of myosin when S1 is bound in rigor (right). Note the azimuthal movement of Tm across F-actin. (C) Projected and enlarged views highlight vital electrostatic interactions of actin residues R147, K326, and K328 with E181 of Tm, in the absence of myosin, and of K328 of actin and E286 of S1 when myosin is bound in rigor. These associations are likely critical for thin filament and muscle function.

Confirmation of transgenic actin transcription. Sequence chromatograms of an amplified stretch of Act57B cDNA revealed transcription of the UAS-Act57B transgenes in the thoracic musculature of Mef2-GAL4> UAS-Act57B transgenic flies. The chromatograms also confirmed the presence and expression of K326Q, K328Q, or K326Q/K328Q actin mutations (identified by the AAG → CAG nucleotide transversion) in the sequenced Act57B cDNA fragments.

Confirmation of muscle-restricted gene expression. Virgin female flies expressing either the muscle-specific MHC-GAL4 or the Mef2-GAL4 driver were mated with male flies carrying the UAS-Act57BGFP.WT construct. Background fluorescence coming from the musculature of the parental lines was minimal. However, fluorescence emitted from the musculature of progeny, which inherit both a GAL4 driver and the UAS-construct, was readily observed in both genotypes, confirming tissue-specific expression of transgenic Act57B actin.

Mef2-GAL4 drives higher expression levels of transgenic actin relative to MHC-GAL4. Quantitative western blot analysis of steady-state Act57BGFP.WT and total actin was performed on thoraces and IFMs of Mef2-GAL4> UAS-Act57BGFP.WT and MHC-GAL4> UAS-Act57BGFP.WT flies and of control flies two days after eclosion. (A) Representative western blots showing elevated thoracic (left) and IFM (right) levels of Act57BGFP.WT (probed with an anti-GFP primary antibody) when driven by Mef2-GAL4 compared to MHC-GAL4. Actin and GAPDH (probed with anti-actin and anti-GAPDH antibodies) abundance appeared relatively consistent among genotypes. The GFP (B) and actin (C) intensities were measured, normalized to that of GAPDH for five thoracic samples with six technical replicates each and for eight IFM samples with four technical replicates each, and averaged for each genotype. Mef2-GAL4 drove significantly higher amounts of transgenic actin relative to MHC-GAL4 (*P < 0.05).